Our long term goal is to understand how a cell builds the mitotic spindle necessary for chromosome movement. We have concentrated efforts on understanding how the cell regulates the microtubule assembly necessary for the construction of this structure. The proposed work focuses on several proteins which are likely key components regulating microtubule assembly: oncoprotein 18/stathmin (Op18), XMAP215, and a human protein related to XMAP215, called TOGp. Both Op18 and TOGp are over-expressed in human tumors but the consequences of over-expression are not known. Proteins related to XMAP215 and TOGp have been identified in yeasts and C. elegans; here mutations support the idea that this protein family functions to regulate both microtubule assembly and chromosome movement. Future studies of Op18, XMAP215 and TOGp will provide important basic information on microtubule assembly in vivo. Since many chemotherapies are based on halting or slowing cell division, a better understanding of mitotic mechanisms has considerable potential health benefits. The specific aim of the current proposal comprise structure/function studies to determine: (1) the domains in Op18 responsible for functional activities; (2) whether Op18 interacts directly with microtubules to promote microtubule turnover; (3) the structure of microtubules assembled with XMAP215 and the orientation of XMAP215 on the microtubule lattice; (4) the microtubule binding domains and assembly-promoting domains within TOGp; and (5) the contributions of XMAP215 and TOGp to rapid microtubule assembly in the cell. For aim 1, we will use in vitro assays to determine the effects of Op18 truncations on microtubule assembly, tubulin GTPase activity and binding to alpha or beta tubulin. We will also probe the activities of selected truncations in living cells. We will use probabilistic analysis to determine whether Op18 modifies the kinetic steps responsible for switches between microtubule growth and shortening and determine whether Op18 stimulates GTP hydrolysis within microtubules (Aim 2). Aim 3 will use microtubule co-pelleting assays, electron microscopy and domain specific antibodies to determine the structure of microtubules bound by XMAP215. For aim 4, fragments of TOGp will be used in in vitro assays to map functional domains. Lastly, for aim 5 we will probe XMAP215 and TOGp functions in vivo by either blocking protein function with antibodies or by increasing protein levels by overexpression. Changes in microtubule turnover will be measured by tubulin fluorescence photoactivation and redistribution.